Cloning, expression and characterization of a novel form of phosphatidylinositol-3-kinase

ABSTRACT

The present invention concerns a new phosphatidyl-inositol-3-kinase (PI3Kγ), a nucleic acid coding for it, an antibody directed against the protein as well as the diagnostic and therapeutic use of the protein, the nucleic acid and the antibody.

RELATED APPLICATION

This application is a national stage application of PCT/EP95/03990 filed Oct. 10, 1995 under 35 USC 371.

BACKGROUND OF THE INVENTION

The present invention concerns a new phosphatidyl-inositol-3-kinase (PI3Kγ), a nucleic acid which codes for it, an antibody directed against the protein as well as the diagnostic and therapeutic use of the protein, of the nucleic acid and of the antibody.

Phosphatidylinositol-kinases belong, together with specific phospholipases, to an enzyme group which catalyses the formation of intracellular messenger substances from the membrane lipid phosphatidyl inositol (PI). The activity of these enzymes is regulated by extracellular effectors such as hormones, growth factors and neurotransmitters. It is assumed that the PI dependent messenger substances are involved among other processes in the regulation of important cells functions such as cell proliferation, secretion of cellular constituents, endocytotic processes, the targetted movement of certain cells, controlled changes of the cytoskeleton. Correspondingly the physiological importance of the PI kinases and phospholipases correlates with a series of disease states involving changes of the functions of these enzymes.

From various experimental results it is possible to conclude that the product of the reaction catalysed by PI3 kinase, PI-3,4,5 triphosphate plays an important role in the regulation of the following physiological cell functions:

Regulation of cell proliferation and cell differentiation by PI 3 kinase

Mitogens such as growth factors and cytokines generally lead to a stimulation of the PI 3 kinase activity in cells capable of division. The oncogenic transformation of cells is also often accompanied by an increase in the measurable PI 3 kinase activity (Varticovski et al., Biochim. Biophys. Acta 1226, 1-11 (1994), Berggren et al., Cancer Research 53, 4297-4302 (1993), Soldi et al., Oncogene 9, 2253-2260 (1994)). Inhibitors of PI 3 kinase are able to inhibit the PDGF-stimulated growth of normal connective tissue cells or smooth muscle cells and the proliferation of src-supratransformed fibroblasts (cancer cells) (Berggren et al., Cancer Research 53, 4297-4302 (1993), Vlahos et al., J.Biol.Chem. 269, 5241-5248 (1994)). Berggren et al., have speculated that the tumoristatic effect of ether lipid analogues is mainly based on their inhibitory action on PI 3 kinase.

The differentiation of the nerve cell line PC12 is suppressed by wortmannin an inhibitor of PI 3 kinase (Kimurea et al., J.Biol.Chem. 269, 18961-18967 (1994)). These findings as well as a clinical study in which there was shown to be a selective loss of PI 3 kinase activity in the brain of Alzheimer patients (Bothmer et al., Dementia 5, 6-11 (1994)) indicate that the enzyme has an important function in the formation and maintenance of nerve tissue.

Regulation of cytoskeletal-dependent processes by PI 3 kinase

Microscopically visible changes of cells often progress with the involvement of the cytoskeleton. A series of results shows that at least some of these processes are regulated by PI 3 kinase and its enzymatic products (PI3,4,5,P₃, PI3,4P₂ and PI3P). Thus the membrane ruffling of epidermal cells induced by insulin or PDGF can be suppressed by the PI 3 kinase inhibitor wortmannin (Kotani et al., EMBO J. 13, 2313-2321 (1994), Wennstrom et al., Curr. Biol. 4, 385-393 (1994)).

Basophilic leucocytes are able to secrete histamine--a mediator of inflammations and allergic symptoms. The cytoskeleton of the cells is involved in this secretion process. Yano et al., J. Biol.Chem. 268, 25846-25856 (1993) were able to show that the antibody-induced histamine secretion can in turn be inhibited by the PI 3 kinase inhibitor wortmannin i.e. it is apparently controlled by 3-phosphorylated phosphoinositides.

Involvement of PI 3 kinase in intracellular transport processes

Investigations on yeast mutants show that one form of PI 3 kinase (Vps 34) is involved in these organisms in the selective distribution of proteins towards the yeast vacuoles (Schu et al., Science 260, 88-91 (1993)). Similar mechanisms may be the basis of the insulin-stimulated translocation of glucose transport protein (GLUT 4) from the interior of the cell to the plasma membrane (Kanai et al., Biochem.Biophys.Res.Commun. 195, 762-768 (1993)). This important process in various organs is also inhibited by wortmannin and apparently involves PI 3 kinase.

Inhibition of the O₂ ⁻ production in neutrophilic granulocytes

Granulocytes produce superoxide anions (O₂ ⁻ ) with the aim of destroying phagocytised foreign cells. This process is stimulated by the chemoattractant fMLP. The blocking of the fMLP-induced O₂ ⁻ formation by wortmannin indicates a regulatory function of a PI 3 kinase species in this important process for the immune response of the body.

The above-mentioned results underlin e the central importance of PI 3 kinase and 3-phosphorylated inositol lipids in the regulation of important cell functions. Acquired or inherited defects of the said cell functions are undoubtedly the underlying cause of important clinical syndromes. Examples are: cancer, arteriosclerosis, immunopathies, skin diseases (such as psoriasis), degenerative diseases of the nervous system.

Since Pi 3 kinase is an essential element in the regulation of the said cell functions it is very probable that some of the clinical syndromes are due to malfunctions of PI 3 kinase species. The clinical study on PI 3 kinase in the brain of Alzheimer patients, the findings on the role of PI 3 kinase in the formation of the allergy inducer histamine and also the cancerostatic effect of PI 3 kinase-inhibiting ether lipids point in this direction.

A central concern of cell biology is to discover the mechanisms of intracellular signal transmission and the messenger substances that are involved. The final goal of these investigations is to selectively influence cell functions in a medical sense.

SUMMARY OF THE INVENTION

The present application describes the identification, cloning, expression and c harac terization of a new species of PI 3 kinase. This new species is activated by G protein subunits and hence differs from the previous species PI3Kα (Hiles et al., Cell 70 (1992) 419-429) and PI3Kβ (Hu et al., J.Mol.Cell. Biol. 13, (1993), 7677-7688) cloned from mammalian cells and from the PI3K-Vps34 from yeast (Schu et al., supra and Herman et al., Cell 64 (1991), 425-438) with regard to the regulation mechanism. The functional differences between the known enzymes PI3Kα and PI3Kβ on the one hand as well as the enzyme PI3Kγ according to the invention on the other hand are reflected in differences in the sequences of the important regulatory domains of the enzyme.

Hence a subject matter of the invention is a protein with phosphatidylinositol-3-kinase activity which is characterized in that it comprises

a) the amino acid sequence shown in SEQ ID NO. 2

b) the amino acid sequence shown in SEQ ID NO. 4 or

c) variants of the sequence from (a).

The PI3Kγ according to the invention is preferably a protein obtainable from humans i.e. it is the protein shown in SEQ ID NO. 1 and No. 2, the protein shown in SEQ ID NO. 3 and NO. 4 or a naturally occurring human variant thereof.

The invention also concerns a new protein which comprises parts of the amino acid sequence shown in SEQ ID NO. 1 and 2 or SEQ ID NO. 3 and 4. The invention preferably concerns a PI3K which comprises the amino acid sequence shown in SEQ ID NO. 1 and 2 or the amino acid sequence shown in SEQ ID NO. 3 and 4; it can, however, also contain variants of this sequence. The term "variant" within the sense of the present invention is understood as sequences which differ from the amino acid sequence shown in SEQ ID NO. 1 and 2 or in SEQ ID NO. 3 and 4 by substitution, deletion or/and insertion of individual amino acids or short sections of amino acids.

The term "variant" includes naturally occurring allelic variations of PI3Kγ as well as proteins produced by recombinant DNA technology (in particular by in vitro mutagenesis with the aid of chemically synthesized oligonucleotides) which, with regard to their biological or/and immunological activity, essentially correspond to the protein shown in SEQ ID NO. 1 and 2 or to the protein shown in SEQ ID NO. 3 and 4.

A preferred characteristic of proteins according to the invention is that at the amino acid level they have a homology of at least 80% particularly preferably of 90% and most preferably of 95% to the amino acid sequence shown in SEQ ID NO. 1 and 2 or to the amino acid sequences shown in SEQ ID NO. 3 and 4.

The amino acid sequence shown in SEQ ID NO. 1 and 2 represents a whole PI3Kγ. This protein has 1049 amino acids and has a molecular mass of ca. 120 kDa. The amino acid sequence shown in SEQ ID NO. 3 and 4 represents a PI3Kγ with 1050 amino acids.

The amino acid sequence of PI3Kγ has a homology of 19 to 39% to the sequences of other known PI3K such as human PI3Kα (36% homology), human PI3Kβ (33.5% homology) and PI3KVps34 from yeast (27.7%). The most highly conserved region in this group of enzymes are the 400 C-terminal amino acid residues which presumably contain the kinase domain. There is no significant homology between PI3Kγ and the other enzymes in the amino-terminal region which is known to be responsible for the binding of PI3Kα and β-enzyme subunits to the regulatory and adaptory subunits p85α and p85β (Dhand et al., EMBO J. 13 (1994), 511-521).

PI3Kγ can be detected in the cell as a 110 kDa protein by immunoprecipitation and Western blot analysis of U937 and K562 cells using antipeptide antisera. A Northern blot analysis showed a 5.3 kb long mRNA in several different tissue types.

A further subject matter of the present invention is a nucleic acid which codes for a phosphatidylinositol-3-kinase or parts thereof according to the invention. This nucleic acid can for example be genomic DNA, cDNA or RNA. It is preferably a recombinant DNA molecule.

Another subject matter of the invention is a nucleic acid which contains

a) the coding sequence shown in SEQ ID NO. 1

b) the protein-coding sequence shown in SEQ ID NO. 3

c) a nucleic acid sequence corresponding to the sequence from (a) or (b) within the scope of the degeneracy of the genetic code or

d) a sequence hybridizing with the sequences from (a), (b) and/or (c) under stringent hybridization conditions.

Stringent hybridization conditions within the sense of the present invention are understood as a hybridization which also still occurs after washing at 55° C., preferably at 62° C., particularly preferably at 68° C. in an aqueous low salt buffer (e.g. 0.2×SSC, 0.1% SDS) (see also Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual).

The invention also concerns nucleic acids which contain at least a 20 nucleotide long section of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3. This section preferably has a specific nucleotide sequence for the PI3Kγ. These nucleic acids are especially suitable for the production of therapeutically applicable antisense nucleic acids which are preferably up to 50 nucleotides long.

Yet a further subject matter of the invention is a vector which contains at least one copy of a nucleic acid or a part thereof according to the invention. The vector can be capable of replication in eukaryotes or prokaryotes. It can be a vector which can be integrated into the genome of the host cell e.g. bacteriophage lambda, or a vector which is present extrachromosomally (e.g. a plasmid). The vector according to the invention can be obtained by subcloning the PI3Kγ DNA into a basic vector. Such basic vectors in particular vectors containing the necessary elements for protein expression are familiar to a person skilled in the art.

When a nucleic acid coding for PI3Kγ is cloned it is possible to construct an expression vector which can be made to express in a suitable host cell to form the protein according to the invention. Preferred host cells are microorganisms such as E. coli or yeast and also higher cells (e.g. mammalian or insect cells). Preferred expression vectors are e.g. plasmids, bacteriophage lambda for prokaryotes, yeast vectors or viral vectors for higher cells (e.g. SV40, vaccinia, baculorivuses). With regard to the expression of a nucleic acid coding for PI3Kγ particular reference is made to the methods mentioned in Sambrook et al. (1989) supra.

A specific example of a system suitable for the expression of PI3Kγ is the expression as a GST fusion protein in Sf9-insect cells using baculovectors according to the method described by Davis et al. (Biotechnology 11 (1993), 933-936).

A further subject matter of the present invention is a cell which is transformed with a nucleic acid according to the invention or a vector according to the invention. The cell can be a eukaryotic as well as a prokaryotic cell. Methods for the transformation of cells with nucleic acids are general state of the art and do not therefore need to be elucidated in more detail.

The use of the PI3Kγ protein or fragments of this protein as an immunogen for the production of antibodies is also a subject matter of the present invention. In this case the antibodies can be produced in the usual manner by immunizing experimental animals with the complete PI3Kγ protein or fragments thereof and subsequently isolating the resulting polyclonal antisera. The method of Kohler and Milstein or further developments thereof can be used to obtain monoclonal antibodies in a known manner from the antibody-producing cells of the experimental animals by cell fusion. It is also possible to produce human monoclonal antibodies.

Hence a further subject matter of the present invention is an antibody against a protein with phosphatidyl-inositol-3-kinase activity which is specific for phosphatidylinositol-3-kinase γ and does not exhibit any cross-reaction with other phosphatidylinositol-3-kinases. Such an antibody can for example be obtained by using a PI3Kγ-specific peptide sequence as the immunogen e.g. a peptide sequence which corresponds to the amino acids 741 to 755 of the amino acid sequence shown in SEQ ID NO. 1 and 2 or to the amino acids 742 to 756 of the amino acid sequence shown in SEQ ID NO. 3 and 4.

The provision of PI3 kinase γ, a nucleic acid which codes for it and an antibody which is directed towards it creates the basis for a specific search for effectors of this protein. The target for these substances should be the regulatory domains of the enzyme which are located in the region of the amino acid residues 1 to 700 of the amino acid sequences shown in SEQ ID NO. 1 and 2 or SEQ ID NO. 3 and 4. Substances which, via this region of the protein, have an inhibitory or activating effect on the activity are able to selectively influence the cell functions regulated by PI3Kγ. Consequently they can be used for the treatment of corresponding clinical pictures. In the case of clinical pictures which are due to a loss of PI3Kγ it may be possible to carry out a gene therapy treatment in which a nucleic acid coding for PI3Kγ optionally together with a nucleic acid which codes for the activating G proteins is transferred by means of vectors e.g. viral vectors into the appropriate target tissue.

Moreover the results that have been presented form the basis for a specific diagnosis of diseases which are causally linked to changes in PI3Kγ activity. These investigations can be carried out with the aid of specific nucleic acid probes for tests at the DNA level i.e. at the gene or transcription level or with the aid of antibodies against PI3Kγ for tests at the protein level.

Hence the present invention also concerns a pharmaceutical composition which comprises a PI3Kγ protein, an antibody directed towards it or a nucleic acid which codes for it as the active component optionally together with standard pharmaceutical auxiliary substances, carrier substances, fillers and diluents.

The pharmaceutical composition according to the invention can be used in particular to influence cell proliferation, receptor-mediated signal transmission, the structure of the cell membrane, the secretion of histamines, the differentiation of nerve cells, glucose transport and anti-lipolysis. Furthermore it can also be used in connection with the therapy of Alzheimer's disease.

The invention is elucidated in more detail by the following examples, figures and sequence protocols.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the detection of PI3Kγ in human blood cells by specific antibodies,

FIG. 2a shows the recombinant expression of PI3Kγ in insect cells,

FIG. 2b shows the detection of the enzymatic activity of purified recombinant PI3Kγ,

FIG. 3 shows the absence of interaction between PI3Kγ and the proteins p85α and p85β,

DETAILED DESCRIPTION

SEQ ID NO. 1 shows a nucleic acid sequence which contains genetic information coding for PI3Kγ and

SEQ ID NO. 2 shows the amino acid sequence of a PI3Kγ.

SEQ ID NO: 3 shows a nucleic acid sequence which contains genetic information coding for a further PI3Kγ and

SEQ ID NO. 4 shows the amino acid sequence of a further PI3Kγ.

EXAMPLE 1 Isolation of PI3Kγ cDNA

In order to isolate cDNA sequences which code for new PI3K a human bone marrow cDNA library was screened using the polymerase chain reaction (PCR). For this degenerate oligonucleotide primers were used corresponding to the amino acid sequences KNGDDLR SEQ ID NO:6! and HIDFG SEQ ID NO:7!. A 402 bp long fragment was obtained. This fragment was subcloned and sequenced.

Several overlapping clones from a human U937 cDNA library were isolated using this PCR fragment as a probe. The largest clone contained the nucleic acid sequence shown in SEQ ID NO. 1 with an open reading frame which codes for a protein with 1049 amino acids (SEQ ID NO. 2). This protein named PI3Kγ has a molecular mass of approximately 120 kDa.

A further PI3Kγ sequence which was obtained from a cDNA library is shown in SEQ ID NO. 3 and codes for a protein with 1050 amino acids (SEQ ID NO. 4).

EXAMPLE 2 Detection of PI3Kγ at a protein and transcript level

A polyclonal rabbit antiserum against PI3Kγ was produced by immunization with a 15 amino acid long peptide of the sequence NSQLPESFRVPYDPG SEQ ID NO:5 (corresponding to amino acids 741 to 755 in SEQ ID NO. 2 or amino acids 742 to 756 in SEQ ID NO. 4). The serum was purified by protein A chromatography and affinity chromatography using the peptide antigen coupled to Actigel (Sterogene).

PI3Kγ can be detected as a 110 kDa protein using this antiserum by immunoprecipitation and Western blot analysis of U937 and K562 cells (FIG. 1). The immunoprecipitation and Western blot were carried out according to the method of Hiles et al. (Cell 70 (1992) 419-429). A conjugate of horseradish peroxidase and anti-rabbit antiserum (Sigma, 1:2000 dilution) was used as the secondary antibody. The bound peroxidase was visualized by chemiluminescence.

A Northern blot analysis of human tissue from the pancreas, kidney, skeletal muscle, liver, lung, placenta, brain and heart each showed different concentrations of a 5.3 kb long mRNA.

EXAMPLE 3 Recombinant expression of PI3Kγ

The DNA coding for PI3Kγ was cloned into the vector pAcG2T (Davis et al., Biotechnology 11 (1993), 933-936). The resulting recombinant vector pAcG2T-PI3Kγ contained the PI3Kγ cDNA from codon 4 onwards in a fusion with the glutathione S transferase (GST) gene. Sf9 cells were cotransfected with pAcG2T-PI3Kγ and linearized baculovirus DNA (BaculoGold, Pharmingen). Individual recombinant baculovirus GST-PI3Kγ plaques were purified and amplified. The expression and purification of recombinant protein were carried out using standard protocols (Dhand et al., EMBO J. 13 (1994), 511-521). FIG. 2a shows the expression of recombinant PI3Kγ GST fusion protein or GST alone after fractionation on a SDS polyacrylamide gel. The detection was carried out by staining with coomassie blue.

FIG. 2b shows a detection of the enzymatic activity of purified recombinant PI3Ky. Phosphatidylinositol (PI, lane 1), phosphatidylinositol-4-phosphate (PI4-P, lane 2) and phosphatidylinositol-4,5-diphosphate (PI 4,5-P₂, lane 3) were used as substrates. The test was carried out essentially according to the method of Stephens et al. (Cell 77 (1994), 83-93) but without cholate. 30 μl sonicated lipid vesicles containing 320 μM phosphatidyl ethanolamine, 140 μM phosphatidyl choline, 300 μM phosphatidyl serine, 30 μM sphingomyelin and 320 μM substrate were added to 10 μl enzyme (0.1 ng) and incubated for 8 minutes on ice. The test was started by addition of 10 μl 20 μM ATP containing 10 μCi γ(³² P)ATP and incubated for 15 minutes at room temperature. The extracted lipids were separated and visualized. The identity of the 3-phosphorylated phosphoinositides was confirmed by anion exchange HPLC after deacylation of the lipids (Auger et al., Cell 57 (1989), 167-175).

FIG. 2b shows that the substrates were phosphorylated in the D-3 position of the inositol ring. In addition it was found that PI3Kγ was inhibited by wortmannin at nanomolar concentrations.

EXAMPLE 4 Regulation of PI3Kγ

In order to detect an interaction between the regulatory and adaptory subunits of p85 (p85α and p85β) and PI3Kγ, human PI3Kγ and bovine PI3Kα (Hiles et al., supra) were expressed in Sf9 insect cells according to the process described in example 3 as GST fusion proteins either alone or together with p85α and p85β. After lysis and centrifugation of the cells, glutathione-Sepharose was added to the supernatant in order to bind the GST fusion proteins. The particles were washed and analysed by SDS-PAGE and Western-blot. PI3Kγ was detected using the polyclonal antipeptide antibody described in example 2. Appropriate mixtures of specific monoclonal antibodies (Dhand et al., supra) as a primary antibody and conjugates of horseradish peroxidase and anti-mouse antibodies (Dianova, 1:4000 dilution) as the secondary antibody were used to detect p85α, p85β and bovine PI3Kα.

FIG. 3 shows that PI3Kγ in contrast to PI3Kα does not bind to p85α and p85β subunits. The regulation mechanism of PI3Kγ is therefore different from PI3Kα.

In order to detect an interaction between PI3Kγ and G proteins, purified recombinant human PI3Kγ and bovine PI3Kα was incubated with the transducin species G_(t) βγ. A substantial amplification of the kinase activity of PI3Kγ was found. This stimulation was suppressed by adding GTP-loaded G_(t) α which confirms the specificity of the interaction.

In contrast the enzymatic activity of PI3Kα could not be stimulated by addition of G_(t) βγ. A similar activation of the enzyme was found after addition of Gα in the presence of 20 μM AlCl₃ and 10 mM NaF. The complex Gα-GDP-AlF₄ - acts as an activating species.

The determination of activity was carried out as described in example 3. The G_(t) proteins purified from bovine retina according to the method of Camps et al. (Nature 360 (1992) 684-686) were incubated on ice with the lipid vesicles for 5 min (G_(t) βγ) or for 20 min (G_(t) βγ plus G_(t) α-GTP) before adding the enzyme.

Binding studies with recombinant PI3Kγ showed that the regulator protein Ras in its active GTP-loaded form associates with the enzyme. Ras in its constitutively active version is able to oncogenically transform cells. Hence this is a further indication for the potential importance of PI3Kγ in the regulation of cellular proliferation.

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 7     (2) INFORMATION FOR SEQ ID NO: 1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 4134 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: both     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (vi) ORIGINAL SOURCE:     (A) ORGANISM: Homo sapiens     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION:423..3569     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:     GAATTCGGCACGAGCACTTCCTTCTCGGCTAGATTATCTGAAACTGTTGTCGGTTCTTGA60     GATGATACTACCACCGAATGTCTGTGTTTCATTGTCTAGTCCAACCTGTATTGTGGATAT120     CTACAACGTTCCGGCAATAGTTTTGCAGGTGCATCACATTTTTGTTTTTGTTTTGGGAGG180     AAAAGGGAGGGCACGGCAGCCAGGCTTCATATTCCTACAAGTGCATGCTTCAAGATTACT240     GTACTTACAGTGTTTCCAACATCTTCTCATAAAAGGGGAAAGCTTCATAGCCTCAACCAT300     GAAGGAAACCAGTCGCATAGGGCATGGAGCTGGAGAACTATAAACAGCCCGTGGTGCTGA360     GAGAGGACAACTGCCGAAGGCGCCGGAGGATGAAGCCGCGCAGTGCTGCCAGCCTGTCCT420     CCATGGAGCTCATCCCCATCGAGTTCGTGCTGCCCACCAGCCAGCGC467     MetGluLeuIleProIleGluPheValLeuProThrSerGlnArg     51015     AAATGCAAGAGCCCCGAAACGGCGCTGCTGCACGTGGCCGGCCACGGC515     LysCysLysSerProGluThrAlaLeuLeuHisValAlaGlyHisGly     202530     AACGTGGAGCAGATGAAGGCCCAGGTGTGGCTGCGAGCGCTGGAGACC563     AsnValGluGlnMetLysAlaGlnValTrpLeuArgAlaLeuGluThr     354045     AGCTGGCGCGGACTTCTACCACCGGCTGGGACCGCATCACTTCCTCCT611     SerTrpArgGlyLeuLeuProProAlaGlyThrAlaSerLeuProPro     505560     GCTCTATCAGAAGAAGGGCAGTGGTACGAGATCTACGACAAGTACCAG659     AlaLeuSerGluGluGlyGlnTrpTyrGluIleTyrAspLysTyrGln     657075     GTGGTGCAGACTCTGGACTGCCTGCGCTACTGGAAGGCCACGCACCGG707     ValValGlnThrLeuAspCysLeuArgTyrTrpLysAlaThrHisArg     80859095     AGCCCGGGCCAGATCCACCTGGTGCAGCGGCACCCGCCCTCCGAGGAG755     SerProGlyGlnIleHisLeuValGlnArgHisProProSerGluGlu     100105110     TCCCAAGCCTTCCAGCGGCAGCTCACGGCGCTGATTGGCTATGACGTC803     SerGlnAlaPheGlnArgGlnLeuThrAlaLeuIleGlyTyrAspVal     115120125     ACTGACGTCAGCAACGTGCACGACGATGAGCTGGAGTTCACGCGCCGT851     ThrAspValSerAsnValHisAspAspGluLeuGluPheThrArgArg     130135140     GGCTTGGTGACCCCGCGCATGGCGGAGGTGGCCAGCCGCGACCCCAAG899     GlyLeuValThrProArgMetAlaGluValAlaSerArgAspProLys     145150155     CTCTACGCCATGCACCCGTGGGTGACGTCCAAGCCCCTCCCGGAGTAC947     LeuTyrAlaMetHisProTrpValThrSerLysProLeuProGluTyr     160165170175     CTGTGGAAGAAGATTGCCAACAACTGCATCTTCATCGTCATTCACCGC995     LeuTrpLysLysIleAlaAsnAsnCysIlePheIleValIleHisArg     180185190     AGCACCACCAGCCAGACCATTAAGGTCTCACCCGACGACACCCCCGGC1043     SerThrThrSerGlnThrIleLysValSerProAspAspThrProGly     195200205     GCCATCCTGCAGAGCTTCTTCACCAAGATGGCCAAGAAGAAATCTCTG1091     AlaIleLeuGlnSerPhePheThrLysMetAlaLysLysLysSerLeu     210215220     ATGGATATTCCCGAAAGCCAAAGCGAACAGGATTTTGTGCTGCGCGTC1139     MetAspIleProGluSerGlnSerGluGlnAspPheValLeuArgVal     225230235     TGTGGCCGGGATGAGTACCTGGTGGGCGAAACGCCCATCAAAAACTTC1187     CysGlyArgAspGluTyrLeuValGlyGluThrProIleLysAsnPhe     240245250255     CAGTGGGTGAGGCACTGCCTCAAGAACGGAGAAGAGATTCACGTGGTA1235     GlnTrpValArgHisCysLeuLysAsnGlyGluGluIleHisValVal     260265270     CTGGACACGCCTCCAGACCCGGCCCTAGACGAGGTGAGGAAGGAAGAG1283     LeuAspThrProProAspProAlaLeuAspGluValArgLysGluGlu     275280285     TGGCCGCTGGTGGACGACTGCACGGGAGTCACCGGCTACCATGAGCAG1331     TrpProLeuValAspAspCysThrGlyValThrGlyTyrHisGluGln     290295300     CTTACCATCCACGGCAAGGACCACGAGAGTGTGTTCACCGTGTCCCTG1379     LeuThrIleHisGlyLysAspHisGluSerValPheThrValSerLeu     305310315     TGGGACTGCGACCGCAAGTTCAGGGTCAAGATCAGAGGCATTGATATC1427     TrpAspCysAspArgLysPheArgValLysIleArgGlyIleAspIle     320325330335     CCCGTCCTGCCTCGGAACACCGACCTCACAGTTTTTGTAGAGGCAAAC1475     ProValLeuProArgAsnThrAspLeuThrValPheValGluAlaAsn     340345350     ATCCAGCATGGGCAACAAGTCCTTTGCCAAAGGAGAACCAGCCCCAAA1523     IleGlnHisGlyGlnGlnValLeuCysGlnArgArgThrSerProLys     355360365     CCCTTCACAGAGGAGGTGCTGTGGAATGTGTGGCTTGAGTTCAGTATC1571     ProPheThrGluGluValLeuTrpAsnValTrpLeuGluPheSerIle     370375380     AAAATCAAAGACTTGCCCAAAGGGGCTCTACTGAACCTCCAGATCTAC1619     LysIleLysAspLeuProLysGlyAlaLeuLeuAsnLeuGlnIleTyr     385390395     TGCGGTAAAGCTCCAGCACTGTCCAGCAAGGCCTCTGCAGAGTCCCCC1667     CysGlyLysAlaProAlaLeuSerSerLysAlaSerAlaGluSerPro     400405410415     AGTTCTGAGTCCAAGGGCAAAGTTCGGCTTCTCTATTATGTGAACCTG1715     SerSerGluSerLysGlyLysValArgLeuLeuTyrTyrValAsnLeu     420425430     CTGCTGATAGACCACCGTTTCCTCCTGCGCCGTGGAGAATACGTCCTC1763     LeuLeuIleAspHisArgPheLeuLeuArgArgGlyGluTyrValLeu     435440445     CACATGTGGCAGATATCTGGGAAGGGAGAAGACCAAGGAAGCTTCAAT1811     HisMetTrpGlnIleSerGlyLysGlyGluAspGlnGlySerPheAsn     450455460     GCTGACAAACTCACGTCTGCAACTAACCCAGACAAGGAGAACTCAATG1859     AlaAspLysLeuThrSerAlaThrAsnProAspLysGluAsnSerMet     465470475     TCCATCTCCATTCTTCTGGACAATTACTGCCACCCGATAGCCCTGCCT1907     SerIleSerIleLeuLeuAspAsnTyrCysHisProIleAlaLeuPro     480485490495     AAGCATCAGCCCACCCCTGACCCGGAAGGGGACCGGGTTCGAGCAGAA1955     LysHisGlnProThrProAspProGluGlyAspArgValArgAlaGlu     500505510     ATGCCCAACCAGCTTCGCAAGCAATTGGAGGCGATCATAGCCACTGAT2003     MetProAsnGlnLeuArgLysGlnLeuGluAlaIleIleAlaThrAsp     515520525     CCACTTAACCCTCTCACAGCAGAGGACAAAGAATTGCTCTGGCATTTT2051     ProLeuAsnProLeuThrAlaGluAspLysGluLeuLeuTrpHisPhe     530535540     AGATACGAAAGCCTTAAGCACCCAAAAGCATATCCTAAGCTATTTAGT2099     ArgTyrGluSerLeuLysHisProLysAlaTyrProLysLeuPheSer     545550555     TCAGTGAAATGGGGACAGCAAGAAATTGTGGCCAAAACATACCAATTG2147     SerValLysTrpGlyGlnGlnGluIleValAlaLysThrTyrGlnLeu     560565570575     TTGGCCAGAAGGGAAGTCTGGGATCAAAGTGCTTTGGATGTTGGGTTA2195     LeuAlaArgArgGluValTrpAspGlnSerAlaLeuAspValGlyLeu     580585590     ACAATGCAGCTCCTGGACTGCAACTTCTCAGATGAAAATGTAAGAGCC2243     ThrMetGlnLeuLeuAspCysAsnPheSerAspGluAsnValArgAla     595600605     ATTGCAGTTCAGAAACTGGAGAGCTTGGAGGACGATGATGTTCTGCAT2291     IleAlaValGlnLysLeuGluSerLeuGluAspAspAspValLeuHis     610615620     TACCTTCTACAATTGGTCCAGGCTGTGAAATTTGAACCATACCATGAT2339     TyrLeuLeuGlnLeuValGlnAlaValLysPheGluProTyrHisAsp     625630635     AGCGCCCTTGCCAGATTTCTGCTGAAGCGTGGTTTAAGAAACAAAAGA2387     SerAlaLeuAlaArgPheLeuLeuLysArgGlyLeuArgAsnLysArg     640645650655     ATTGGTCACTTTTTGTTTTGGTTCTTGAGAAGTGAGATAGCCCAGTCC2435     IleGlyHisPheLeuPheTrpPheLeuArgSerGluIleAlaGlnSer     660665670     AGACACTATCAGCAGAGGTTCGCTGTGATTCTGGAAGCCTATCTGAGG2483     ArgHisTyrGlnGlnArgPheAlaValIleLeuGluAlaTyrLeuArg     675680685     GGCTGTGGCACAGCCATGCTGCACGACTTTACCCAACAAGTCCAAGTA2531     GlyCysGlyThrAlaMetLeuHisAspPheThrGlnGlnValGlnVal     690695700     ATCGAGATGTTACAAAAAGTCACCCTTGATATTAAATCGCTCTCTGCT2579     IleGluMetLeuGlnLysValThrLeuAspIleLysSerLeuSerAla     705710715     GAAAAGTATGACGTCAGTTCCCAAGTTATTTCACAACTTAAACAAAAG2627     GluLysTyrAspValSerSerGlnValIleSerGlnLeuLysGlnLys     720725730735     CTTGAAAACCTGCAGAATTCTCAACTCCCCGAAAGCTTTAGAGTTCCA2675     LeuGluAsnLeuGlnAsnSerGlnLeuProGluSerPheArgValPro     740745750     TATGATCCTGGACTGAAAGCAGGAGCGCTGGCAATTGAAAAATGTAAA2723     TyrAspProGlyLeuLysAlaGlyAlaLeuAlaIleGluLysCysLys     755760765     GTAATGGCCTCCAAGAAAAAACCACTATGGCTTGAGTTTAAATGTGCC2771     ValMetAlaSerLysLysLysProLeuTrpLeuGluPheLysCysAla     770775780     GATCCTACAGCCCTATCAAATGAAACAATTGGAATTATCTTTAAACAT2819     AspProThrAlaLeuSerAsnGluThrIleGlyIleIlePheLysHis     785790795     GGTGATGATCTGCGCCAAGACATGCTTATTTTACAGATTCTACGAATC2867     GlyAspAspLeuArgGlnAspMetLeuIleLeuGlnIleLeuArgIle     800805810815     ATGGAGTCTATTTGGGAGACTGAATCTTTGGATCTATGCCTCCTGCCA2915     MetGluSerIleTrpGluThrGluSerLeuAspLeuCysLeuLeuPro     820825830     TATGGTTGCATTTCAACTGGTGACAAAATAGGAATGATCGAGATTGTG2963     TyrGlyCysIleSerThrGlyAspLysIleGlyMetIleGluIleVal     835840845     AAAGACGCCACGACAATTGCCAAAATTCAGCAAAGCACAGTGGGCAAC3011     LysAspAlaThrThrIleAlaLysIleGlnGlnSerThrValGlyAsn     850855860     ACGGGAGCATTTAAAGATGAAGTCCTGAATCACTGGCTCAAAGAAAAA3059     ThrGlyAlaPheLysAspGluValLeuAsnHisTrpLeuLysGluLys     865870875     TCCCCTACTGAAGAAAAGTTTCAGGCAGCAGTGGAGAGATTTGTTTAT3107     SerProThrGluGluLysPheGlnAlaAlaValGluArgPheValTyr     880885890895     TCCTGTGCAGGCTACTGTGTGGCAACCTTTGTTCTTGGAATAGGCGAC3155     SerCysAlaGlyTyrCysValAlaThrPheValLeuGlyIleGlyAsp     900905910     AGACACAATGACAATATTATGATCACCGAGACAGGAAACCTATTTCAT3203     ArgHisAsnAspAsnIleMetIleThrGluThrGlyAsnLeuPheHis     915920925     ATTGACTTCGGGCACATTCTTGGGAATTACAAAAGTTTCCTGGGCATT3251     IleAspPheGlyHisIleLeuGlyAsnTyrLysSerPheLeuGlyIle     930935940     AATAAAGAGAGAGTGCCATTTGTGCTAACCCCTGACTTCCTCTTTGTG3299     AsnLysGluArgValProPheValLeuThrProAspPheLeuPheVal     945950955     ATGGGAACTTCTGGAAAGAAGACAAGCCCACACTTCCAGAAATTTCAG3347     MetGlyThrSerGlyLysLysThrSerProHisPheGlnLysPheGln     960965970975     GACATCTGTGTTAAGGCTTATCTAGCCCTTCGTCATCACACAAACCTA3395     AspIleCysValLysAlaTyrLeuAlaLeuArgHisHisThrAsnLeu     980985990     CTGATCATCCTGTTCTCCATGATGCTGATGACAGGAATGCCCCAGTTA3443     LeuIleIleLeuPheSerMetMetLeuMetThrGlyMetProGlnLeu     99510001005     ACAAGCAAAGAAGACATTGAATATATCCGGGATGCCCTCACAGTGGGG3491     ThrSerLysGluAspIleGluTyrIleArgAspAlaLeuThrValGly     101010151020     AAAAATGAGGAGGATGCTAAAAAGTATTTTCTTGATCAGATCGAAGTT3539     LysAsnGluGluAspAlaLysLysTyrPheLeuAspGlnIleGluVal     102510301035     TGGCAGAGACAAAGGATGGACTGTGCAGTTTAATTGGTTTCTACATCTTG3589     TrpGlnArgGlnArgMetAspCysAlaVal     10401045     TTCTTGGCATCAAACAAGGAGAGAAACATTCAGCCTAATACTTTAGGCTAGAATCAAAAA3649     CAAGTTAGTGTTCTATGGTTTAAATTAGCATAGCAATCATCGAACTTGGATTTCAAATGC3709     AATAGACATTGTGAAAGCTGGCATTTCAGAAGTATAGCTCTTTTCCTACCTGAACTCTTC3769     CCTGGAGAAAAGATGTTGGCATTGCTGATTGTTTGGTTAAGCAATGTCCAGTGCTAGGAT3829     TATTTGCAGGTTTGGTTTTTTCTCATTTGTCTGTGGCATTGGAGAATATTCTCGGTTTAA3889     ACAGACTAATGACTTCCTTATTGTCCCTGATATTTTGACTATCTTACTATTGAGTGCTTC3949     TGGAAATTCTTTGGAATAATTGATGACATCTATTTTCATCTGGGTTTAGTCTCAATTTTG4009     GTTATCTTTGTGTTCCTCAAGCTCTTTAAAGAAAAAGATGTAATCGTTGTAACCTTTGTC4069     TCATTCCTTAAATGATGCTTCCAAACATCTCCTTAGTGTCTGCAGGTGTTAGTGGTGTGC4129     TAAAA4134     (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1049 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:     MetGluLeuIleProIleGluPheValLeuProThrSerGlnArgLys     151015     CysLysSerProGluThrAlaLeuLeuHisValAlaGlyHisGlyAsn     202530     ValGluGlnMetLysAlaGlnValTrpLeuArgAlaLeuGluThrSer     354045     TrpArgGlyLeuLeuProProAlaGlyThrAlaSerLeuProProAla     505560     LeuSerGluGluGlyGlnTrpTyrGluIleTyrAspLysTyrGlnVal     65707580     ValGlnThrLeuAspCysLeuArgTyrTrpLysAlaThrHisArgSer     859095     ProGlyGlnIleHisLeuValGlnArgHisProProSerGluGluSer     100105110     GlnAlaPheGlnArgGlnLeuThrAlaLeuIleGlyTyrAspValThr     115120125     AspValSerAsnValHisAspAspGluLeuGluPheThrArgArgGly     130135140     LeuValThrProArgMetAlaGluValAlaSerArgAspProLysLeu     145150155160     TyrAlaMetHisProTrpValThrSerLysProLeuProGluTyrLeu     165170175     TrpLysLysIleAlaAsnAsnCysIlePheIleValIleHisArgSer     180185190     ThrThrSerGlnThrIleLysValSerProAspAspThrProGlyAla     195200205     IleLeuGlnSerPhePheThrLysMetAlaLysLysLysSerLeuMet     210215220     AspIleProGluSerGlnSerGluGlnAspPheValLeuArgValCys     225230235240     GlyArgAspGluTyrLeuValGlyGluThrProIleLysAsnPheGln     245250255     TrpValArgHisCysLeuLysAsnGlyGluGluIleHisValValLeu     260265270     AspThrProProAspProAlaLeuAspGluValArgLysGluGluTrp     275280285     ProLeuValAspAspCysThrGlyValThrGlyTyrHisGluGlnLeu     290295300     ThrIleHisGlyLysAspHisGluSerValPheThrValSerLeuTrp     305310315320     AspCysAspArgLysPheArgValLysIleArgGlyIleAspIlePro     325330335     ValLeuProArgAsnThrAspLeuThrValPheValGluAlaAsnIle     340345350     GlnHisGlyGlnGlnValLeuCysGlnArgArgThrSerProLysPro     355360365     PheThrGluGluValLeuTrpAsnValTrpLeuGluPheSerIleLys     370375380     IleLysAspLeuProLysGlyAlaLeuLeuAsnLeuGlnIleTyrCys     385390395400     GlyLysAlaProAlaLeuSerSerLysAlaSerAlaGluSerProSer     405410415     SerGluSerLysGlyLysValArgLeuLeuTyrTyrValAsnLeuLeu     420425430     LeuIleAspHisArgPheLeuLeuArgArgGlyGluTyrValLeuHis     435440445     MetTrpGlnIleSerGlyLysGlyGluAspGlnGlySerPheAsnAla     450455460     AspLysLeuThrSerAlaThrAsnProAspLysGluAsnSerMetSer     465470475480     IleSerIleLeuLeuAspAsnTyrCysHisProIleAlaLeuProLys     485490495     HisGlnProThrProAspProGluGlyAspArgValArgAlaGluMet     500505510     ProAsnGlnLeuArgLysGlnLeuGluAlaIleIleAlaThrAspPro     515520525     LeuAsnProLeuThrAlaGluAspLysGluLeuLeuTrpHisPheArg     530535540     TyrGluSerLeuLysHisProLysAlaTyrProLysLeuPheSerSer     545550555560     ValLysTrpGlyGlnGlnGluIleValAlaLysThrTyrGlnLeuLeu     565570575     AlaArgArgGluValTrpAspGlnSerAlaLeuAspValGlyLeuThr     580585590     MetGlnLeuLeuAspCysAsnPheSerAspGluAsnValArgAlaIle     595600605     AlaValGlnLysLeuGluSerLeuGluAspAspAspValLeuHisTyr     610615620     LeuLeuGlnLeuValGlnAlaValLysPheGluProTyrHisAspSer     625630635640     AlaLeuAlaArgPheLeuLeuLysArgGlyLeuArgAsnLysArgIle     645650655     GlyHisPheLeuPheTrpPheLeuArgSerGluIleAlaGlnSerArg     660665670     HisTyrGlnGlnArgPheAlaValIleLeuGluAlaTyrLeuArgGly     675680685     CysGlyThrAlaMetLeuHisAspPheThrGlnGlnValGlnValIle     690695700     GluMetLeuGlnLysValThrLeuAspIleLysSerLeuSerAlaGlu     705710715720     LysTyrAspValSerSerGlnValIleSerGlnLeuLysGlnLysLeu     725730735     GluAsnLeuGlnAsnSerGlnLeuProGluSerPheArgValProTyr     740745750     AspProGlyLeuLysAlaGlyAlaLeuAlaIleGluLysCysLysVal     755760765     MetAlaSerLysLysLysProLeuTrpLeuGluPheLysCysAlaAsp     770775780     ProThrAlaLeuSerAsnGluThrIleGlyIleIlePheLysHisGly     785790795800     AspAspLeuArgGlnAspMetLeuIleLeuGlnIleLeuArgIleMet     805810815     GluSerIleTrpGluThrGluSerLeuAspLeuCysLeuLeuProTyr     820825830     GlyCysIleSerThrGlyAspLysIleGlyMetIleGluIleValLys     835840845     AspAlaThrThrIleAlaLysIleGlnGlnSerThrValGlyAsnThr     850855860     GlyAlaPheLysAspGluValLeuAsnHisTrpLeuLysGluLysSer     865870875880     ProThrGluGluLysPheGlnAlaAlaValGluArgPheValTyrSer     885890895     CysAlaGlyTyrCysValAlaThrPheValLeuGlyIleGlyAspArg     900905910     HisAsnAspAsnIleMetIleThrGluThrGlyAsnLeuPheHisIle     915920925     AspPheGlyHisIleLeuGlyAsnTyrLysSerPheLeuGlyIleAsn     930935940     LysGluArgValProPheValLeuThrProAspPheLeuPheValMet     945950955960     GlyThrSerGlyLysLysThrSerProHisPheGlnLysPheGlnAsp     965970975     IleCysValLysAlaTyrLeuAlaLeuArgHisHisThrAsnLeuLeu     980985990     IleIleLeuPheSerMetMetLeuMetThrGlyMetProGlnLeuThr     99510001005     SerLysGluAspIleGluTyrIleArgAspAlaLeuThrValGlyLys     101010151020     AsnGluGluAspAlaLysLysTyrPheLeuAspGlnIleGluValTrp     1025103010351040     GlnArgGlnArgMetAspCysAlaVal     1045     (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 4137 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: both     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (vi) ORIGINAL SOURCE:     (A) ORGANISM: Homo sapiens     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION:423..3572     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:     GAATTCGGCACGAGCACTTCCTTCTCGGCTAGATTATCTGAAACTGTTGTCGGTTCTTGA60     GATGATACTACCACCGAATGTCTGTGTTTCATTGTCTAGTCCAACCTGTATTGTGGATAT120     CTACAACGTTCCGGCAATAGTTTTGCAGGTGCATCACATTTTTGTTTTTGTTTTGGGAGG180     AAAAGGGAGGGCACGGCAGCCAGGCTTCATATTCCTACAAGTGCATGCTTCAAGATTACT240     GTACTTACAGTGTTTCCAACATCTTCTCATAAAAGGGGAAAGCTTCATAGCCTCAACCAT300     GAAGGAAACCAGTCGCATAGGGCATGGAGCTGGAGAACTATAAACAGCCCGTGGTGCTGA360     GAGAGGACAACTGCCGAAGGCGCCGGAGGATGAAGCCGCGCAGTGCTGCCAGCCTGTCCT420     CCATGGAGCTCATCCCCATCGAGTTCGTGCTGCCCACCAGCCAGCGC467     MetGluLeuIleProIleGluPheValLeuProThrSerGlnArg     105010551060     AAATGCAAGAGCCCCGAAACGGCGCTGCTGCACGTGGCCGGCCACGGC515     LysCysLysSerProGluThrAlaLeuLeuHisValAlaGlyHisGly     1065107010751080     AACGTGGAGCAGATGAAGGCCCAGGTGTGGCTGCGAGCGCTGGAGACC563     AsnValGluGlnMetLysAlaGlnValTrpLeuArgAlaLeuGluThr     108510901095     AGCGTGGCGGCGGACTTCTACCACCGGCTGGGACCGCATCACTTCCTC611     SerValAlaAlaAspPheTyrHisArgLeuGlyProHisHisPheLeu     110011051110     CTGCTCTATCAGAAGAAGGGGCAGTGGTACGAGATCTACGACAAGTAC659     LeuLeuTyrGlnLysLysGlyGlnTrpTyrGluIleTyrAspLysTyr     111511201125     CAGGTGGTGCAGACTCTGGACTGCCTGCGCTACTGGAAGGCCACGCAC707     GlnValValGlnThrLeuAspCysLeuArgTyrTrpLysAlaThrHis     113011351140     CGGAGCCCGGGCCAGATCCACCTGGTGCAGCGGCACCCGCCCTCCGAG755     ArgSerProGlyGlnIleHisLeuValGlnArgHisProProSerGlu     1145115011551160     GAGTCCCAAGCCTTCCAGCGGCAGCTCACGGCGCTGATTGGCTATGAC803     GluSerGlnAlaPheGlnArgGlnLeuThrAlaLeuIleGlyTyrAsp     116511701175     GTCACTGACGTCAGCAACGTGCACGACGATGAGCTGGAGTTCACGCGC851     ValThrAspValSerAsnValHisAspAspGluLeuGluPheThrArg     118011851190     CGTGGCTTGGTGACCCCGCGCATGGCGGAGGTGGCCAGCCGCGACCCC899     ArgGlyLeuValThrProArgMetAlaGluValAlaSerArgAspPro     119512001205     AAGCTCTACGCCATGCACCCGTGGGTGACGTCCAAGCCCCTCCCGGAG947     LysLeuTyrAlaMetHisProTrpValThrSerLysProLeuProGlu     121012151220     TACCTGTGGAAGAAGATTGCCAACAACTGCATCTTCATCGTCATTCAC995     TyrLeuTrpLysLysIleAlaAsnAsnCysIlePheIleValIleHis     1225123012351240     CGCAGCACCACCAGCCAGACCATTAAGGTCTCACCCGACGACACCCCC1043     ArgSerThrThrSerGlnThrIleLysValSerProAspAspThrPro     124512501255     GGCGCCATCCTGCAGAGCTTCTTCACCAAGATGGCCAAGAAGAAATCT1091     GlyAlaIleLeuGlnSerPhePheThrLysMetAlaLysLysLysSer     126012651270     CTGATGGATATTCCCGAAAGCCAAAGCGAACAGGATTTTGTGCTGCGC1139     LeuMetAspIleProGluSerGlnSerGluGlnAspPheValLeuArg     127512801285     GTCTGTGGCCGGGATGAGTACCTGGTGGGCGAAACGCCCATCAAAAAC1187     ValCysGlyArgAspGluTyrLeuValGlyGluThrProIleLysAsn     129012951300     TTCCAGTGGGTGAGGCACTGCCTCAAGAACGGAGAAGAGATTCACGTG1235     PheGlnTrpValArgHisCysLeuLysAsnGlyGluGluIleHisVal     1305131013151320     GTACTGGACACGCCTCCAGACCCGGCCCTAGACGAGGTGAGGAAGGAA1283     ValLeuAspThrProProAspProAlaLeuAspGluValArgLysGlu     132513301335     GAGTGGCCGCTGGTGGACGACTGCACGGGAGTCACCGGCTACCATGAG1331     GluTrpProLeuValAspAspCysThrGlyValThrGlyTyrHisGlu     134013451350     CAGCTTACCATCCACGGCAAGGACCACGAGAGTGTGTTCACCGTGTCC1379     GlnLeuThrIleHisGlyLysAspHisGluSerValPheThrValSer     135513601365     CTGTGGGACTGCGACCGCAAGTTCAGGGTCAAGATCAGAGGCATTGAT1427     LeuTrpAspCysAspArgLysPheArgValLysIleArgGlyIleAsp     137013751380     ATCCCCGTCCTGCCTCGGAACACCGACCTCACAGTTTTTGTAGAGGCA1475     IleProValLeuProArgAsnThrAspLeuThrValPheValGluAla     1385139013951400     AACATCCAGCATGGGCAACAAGTCCTTTGCCAAAGGAGAACCAGCCCC1523     AsnIleGlnHisGlyGlnGlnValLeuCysGlnArgArgThrSerPro     140514101415     AAACCCTTCACAGAGGAGGTGCTGTGGAATGTGTGGCTTGAGTTCAGT1571     LysProPheThrGluGluValLeuTrpAsnValTrpLeuGluPheSer     142014251430     ATCAAAATCAAAGACTTGCCCAAAGGGGCTCTACTGAACCTCCAGATC1619     IleLysIleLysAspLeuProLysGlyAlaLeuLeuAsnLeuGlnIle     143514401445     TACTGCGGTAAAGCTCCAGCACTGTCCAGCAAGGCCTCTGCAGAGTCC1667     TyrCysGlyLysAlaProAlaLeuSerSerLysAlaSerAlaGluSer     145014551460     CCCAGTTCTGAGTCCAAGGGCAAAGTTCGGCTTCTCTATTATGTGAAC1715     ProSerSerGluSerLysGlyLysValArgLeuLeuTyrTyrValAsn     1465147014751480     CTGCTGCTGATAGACCACCGTTTCCTCCTGCGCCGTGGAGAATACGTC1763     LeuLeuLeuIleAspHisArgPheLeuLeuArgArgGlyGluTyrVal     148514901495     CTCCACATGTGGCAGATATCTGGGAAGGGAGAAGACCAAGGAAGCTTC1811     LeuHisMetTrpGlnIleSerGlyLysGlyGluAspGlnGlySerPhe     150015051510     AATGCTGACAAACTCACGTCTGCAACTAACCCAGACAAGGAGAACTCA1859     AsnAlaAspLysLeuThrSerAlaThrAsnProAspLysGluAsnSer     151515201525     ATGTCCATCTCCATTCTTCTGGACAATTACTGCCACCCGATAGCCCTG1907     MetSerIleSerIleLeuLeuAspAsnTyrCysHisProIleAlaLeu     153015351540     CCTAAGCATCAGCCCACCCCTGACCCGGAAGGGGACCGGGTTCGAGCA1955     ProLysHisGlnProThrProAspProGluGlyAspArgValArgAla     1545155015551560     GAAATGCCCAACCAGCTTCGCAAGCAATTGGAGGCGATCATAGCCACT2003     GluMetProAsnGlnLeuArgLysGlnLeuGluAlaIleIleAlaThr     156515701575     GATCCACTTAACCCTCTCACAGCAGAGGACAAAGAATTGCTCTGGCAT2051     AspProLeuAsnProLeuThrAlaGluAspLysGluLeuLeuTrpHis     158015851590     TTTAGATACGAAAGCCTTAAGCACCCAAAAGCATATCCTAAGCTATTT2099     PheArgTyrGluSerLeuLysHisProLysAlaTyrProLysLeuPhe     159516001605     AGTTCAGTGAAATGGGGACAGCAAGAAATTGTGGCCAAAACATACCAA2147     SerSerValLysTrpGlyGlnGlnGluIleValAlaLysThrTyrGln     161016151620     TTGTTGGCCAGAAGGGAAGTCTGGGATCAAAGTGCTTTGGATGTTGGG2195     LeuLeuAlaArgArgGluValTrpAspGlnSerAlaLeuAspValGly     1625163016351640     TTAACAATGCAGCTCCTGGACTGCAACTTCTCAGATGAAAATGTAAGA2243     LeuThrMetGlnLeuLeuAspCysAsnPheSerAspGluAsnValArg     164516501655     GCCATTGCAGTTCAGAAACTGGAGAGCTTGGAGGACGATGATGTTCTG2291     AlaIleAlaValGlnLysLeuGluSerLeuGluAspAspAspValLeu     166016651670     CATTACCTTCTACAATTGGTCCAGGCTGTGAAATTTGAACCATACCAT2339     HisTyrLeuLeuGlnLeuValGlnAlaValLysPheGluProTyrHis     167516801685     GATAGCGCCCTTGCCAGATTTCTGCTGAAGCGTGGTTTAAGAAACAAA2387     AspSerAlaLeuAlaArgPheLeuLeuLysArgGlyLeuArgAsnLys     169016951700     AGAATTGGTCACTTTTTGTTTTGGTTCTTGAGAAGTGAGATAGCCCAG2435     ArgIleGlyHisPheLeuPheTrpPheLeuArgSerGluIleAlaGln     1705171017151720     TCCAGACACTATCAGCAGAGGTTCGCTGTGATTCTGGAAGCCTATCTG2483     SerArgHisTyrGlnGlnArgPheAlaValIleLeuGluAlaTyrLeu     172517301735     AGGGGCTGTGGCACAGCCATGCTGCACGACTTTACCCAACAAGTCCAA2531     ArgGlyCysGlyThrAlaMetLeuHisAspPheThrGlnGlnValGln     174017451750     GTAATCGAGATGTTACAAAAAGTCACCCTTGATATTAAATCGCTCTCT2579     ValIleGluMetLeuGlnLysValThrLeuAspIleLysSerLeuSer     175517601765     GCTGAAAAGTATGACGTCAGTTCCCAAGTTATTTCACAACTTAAACAA2627     AlaGluLysTyrAspValSerSerGlnValIleSerGlnLeuLysGln     177017751780     AAGCTTGAAAACCTGCAGAATTCTCAACTCCCCGAAAGCTTTAGAGTT2675     LysLeuGluAsnLeuGlnAsnSerGlnLeuProGluSerPheArgVal     1785179017951800     CCATATGATCCTGGACTGAAAGCAGGAGCGCTGGCAATTGAAAAATGT2723     ProTyrAspProGlyLeuLysAlaGlyAlaLeuAlaIleGluLysCys     180518101815     AAAGTAATGGCCTCCAAGAAAAAACCACTATGGCTTGAGTTTAAATGT2771     LysValMetAlaSerLysLysLysProLeuTrpLeuGluPheLysCys     182018251830     GCCGATCCTACAGCCCTATCAAATGAAACAATTGGAATTATCTTTAAA2819     AlaAspProThrAlaLeuSerAsnGluThrIleGlyIleIlePheLys     183518401845     CATGGTGATGATCTGCGCCAAGACATGCTTATTTTACAGATTCTACGA2867     HisGlyAspAspLeuArgGlnAspMetLeuIleLeuGlnIleLeuArg     185018551860     ATCATGGAGTCTATTTGGGAGACTGAATCTTTGGATCTATGCCTCCTG2915     IleMetGluSerIleTrpGluThrGluSerLeuAspLeuCysLeuLeu     1865187018751880     CCATATGGTTGCATTTCAACTGGTGACAAAATAGGAATGATCGAGATT2963     ProTyrGlyCysIleSerThrGlyAspLysIleGlyMetIleGluIle     188518901895     GTGAAAGACGCCACGACAATTGCCAAAATTCAGCAAAGCACAGTGGGC3011     ValLysAspAlaThrThrIleAlaLysIleGlnGlnSerThrValGly     190019051910     AACACGGGAGCATTTAAAGATGAAGTCCTGAATCACTGGCTCAAAGAA3059     AsnThrGlyAlaPheLysAspGluValLeuAsnHisTrpLeuLysGlu     191519201925     AAATCCCCTACTGAAGAAAAGTTTCAGGCAGCAGTGGAGAGATTTGTT3107     LysSerProThrGluGluLysPheGlnAlaAlaValGluArgPheVal     193019351940     TATTCCTGTGCAGGCTACTGTGTGGCAACCTTTGTTCTTGGAATAGGC3155     TyrSerCysAlaGlyTyrCysValAlaThrPheValLeuGlyIleGly     1945195019551960     GACAGACACAATGACAATATTATGATCACCGAGACAGGAAACCTATTT3203     AspArgHisAsnAspAsnIleMetIleThrGluThrGlyAsnLeuPhe     196519701975     CATATTGACTTCGGGCACATTCTTGGGAATTACAAAAGTTTCCTGGGC3251     HisIleAspPheGlyHisIleLeuGlyAsnTyrLysSerPheLeuGly     198019851990     ATTAATAAAGAGAGAGTGCCATTTGTGCTAACCCCTGACTTCCTCTTT3299     IleAsnLysGluArgValProPheValLeuThrProAspPheLeuPhe     199520002005     GTGATGGGAACTTCTGGAAAGAAGACAAGCCCACACTTCCAGAAATTT3347     ValMetGlyThrSerGlyLysLysThrSerProHisPheGlnLysPhe     201020152020     CAGGACATCTGTGTTAAGGCTTATCTAGCCCTTCGTCATCACACAAAC3395     GlnAspIleCysValLysAlaTyrLeuAlaLeuArgHisHisThrAsn     2025203020352040     CTACTGATCATCCTGTTCTCCATGATGCTGATGACAGGAATGCCCCAG3443     LeuLeuIleIleLeuPheSerMetMetLeuMetThrGlyMetProGln     204520502055     TTAACAAGCAAAGAAGACATTGAATATATCCGGGATGCCCTCACAGTG3491     LeuThrSerLysGluAspIleGluTyrIleArgAspAlaLeuThrVal     206020652070     GGGAAAAATGAGGAGGATGCTAAAAAGTATTTTCTTGATCAGATCGAA3539     GlyLysAsnGluGluAspAlaLysLysTyrPheLeuAspGlnIleGlu     207520802085     GTTTGGCAGAGACAAAGGATGGACTGTGCAGTTTAATTGGTTTCTACATCTTG3592     ValTrpGlnArgGlnArgMetAspCysAlaVal     20902095     TTCTTGGCATCAAACAAGGAGAGAAACATTCAGCCTAATACTTTAGGCTAGAATCAAAAA3652     CAAGTTAGTGTTCTATGGTTTAAATTAGCATAGCAATCATCGAACTTGGATTTCAAATGC3712     AATAGACATTGTGAAAGCTGGCATTTCAGAAGTATAGCTCTTTTCCTACCTGAACTCTTC3772     CCTGGAGAAAAGATGTTGGCATTGCTGATTGTTTGGTTAAGCAATGTCCAGTGCTAGGAT3832     TATTTGCAGGTTTGGTTTTTTCTCATTTGTCTGTGGCATTGGAGAATATTCTCGGTTTAA3892     ACAGACTAATGACTTCCTTATTGTCCCTGATATTTTGACTATCTTACTATTGAGTGCTTC3952     TGGAAATTCTTTGGAATAATTGATGACATCTATTTTCATCTGGGTTTAGTCTCAATTTTG4012     GTTATCTTTGTGTTCCTCAAGCTCTTTAAAGAAAAAGATGTAATCGTTGTAACCTTTGTC4072     TCATTCCTTAAATGATGCTTCCAAACATCTCCTTAGTGTCTGCAGGTGTTAGTGGTGTGC4132     TAAAA4137     (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1050 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:     MetGluLeuIleProIleGluPheValLeuProThrSerGlnArgLys     151015     CysLysSerProGluThrAlaLeuLeuHisValAlaGlyHisGlyAsn     202530     ValGluGlnMetLysAlaGlnValTrpLeuArgAlaLeuGluThrSer     354045     ValAlaAlaAspPheTyrHisArgLeuGlyProHisHisPheLeuLeu     505560     LeuTyrGlnLysLysGlyGlnTrpTyrGluIleTyrAspLysTyrGln     65707580     ValValGlnThrLeuAspCysLeuArgTyrTrpLysAlaThrHisArg     859095     SerProGlyGlnIleHisLeuValGlnArgHisProProSerGluGlu     100105110     SerGlnAlaPheGlnArgGlnLeuThrAlaLeuIleGlyTyrAspVal     115120125     ThrAspValSerAsnValHisAspAspGluLeuGluPheThrArgArg     130135140     GlyLeuValThrProArgMetAlaGluValAlaSerArgAspProLys     145150155160     LeuTyrAlaMetHisProTrpValThrSerLysProLeuProGluTyr     165170175     LeuTrpLysLysIleAlaAsnAsnCysIlePheIleValIleHisArg     180185190     SerThrThrSerGlnThrIleLysValSerProAspAspThrProGly     195200205     AlaIleLeuGlnSerPhePheThrLysMetAlaLysLysLysSerLeu     210215220     MetAspIleProGluSerGlnSerGluGlnAspPheValLeuArgVal     225230235240     CysGlyArgAspGluTyrLeuValGlyGluThrProIleLysAsnPhe     245250255     GlnTrpValArgHisCysLeuLysAsnGlyGluGluIleHisValVal     260265270     LeuAspThrProProAspProAlaLeuAspGluValArgLysGluGlu     275280285     TrpProLeuValAspAspCysThrGlyValThrGlyTyrHisGluGln     290295300     LeuThrIleHisGlyLysAspHisGluSerValPheThrValSerLeu     305310315320     TrpAspCysAspArgLysPheArgValLysIleArgGlyIleAspIle     325330335     ProValLeuProArgAsnThrAspLeuThrValPheValGluAlaAsn     340345350     IleGlnHisGlyGlnGlnValLeuCysGlnArgArgThrSerProLys     355360365     ProPheThrGluGluValLeuTrpAsnValTrpLeuGluPheSerIle     370375380     LysIleLysAspLeuProLysGlyAlaLeuLeuAsnLeuGlnIleTyr     385390395400     CysGlyLysAlaProAlaLeuSerSerLysAlaSerAlaGluSerPro     405410415     SerSerGluSerLysGlyLysValArgLeuLeuTyrTyrValAsnLeu     420425430     LeuLeuIleAspHisArgPheLeuLeuArgArgGlyGluTyrValLeu     435440445     HisMetTrpGlnIleSerGlyLysGlyGluAspGlnGlySerPheAsn     450455460     AlaAspLysLeuThrSerAlaThrAsnProAspLysGluAsnSerMet     465470475480     SerIleSerIleLeuLeuAspAsnTyrCysHisProIleAlaLeuPro     485490495     LysHisGlnProThrProAspProGluGlyAspArgValArgAlaGlu     500505510     MetProAsnGlnLeuArgLysGlnLeuGluAlaIleIleAlaThrAsp     515520525     ProLeuAsnProLeuThrAlaGluAspLysGluLeuLeuTrpHisPhe     530535540     ArgTyrGluSerLeuLysHisProLysAlaTyrProLysLeuPheSer     545550555560     SerValLysTrpGlyGlnGlnGluIleValAlaLysThrTyrGlnLeu     565570575     LeuAlaArgArgGluValTrpAspGlnSerAlaLeuAspValGlyLeu     580585590     ThrMetGlnLeuLeuAspCysAsnPheSerAspGluAsnValArgAla     595600605     IleAlaValGlnLysLeuGluSerLeuGluAspAspAspValLeuHis     610615620     TyrLeuLeuGlnLeuValGlnAlaValLysPheGluProTyrHisAsp     625630635640     SerAlaLeuAlaArgPheLeuLeuLysArgGlyLeuArgAsnLysArg     645650655     IleGlyHisPheLeuPheTrpPheLeuArgSerGluIleAlaGlnSer     660665670     ArgHisTyrGlnGlnArgPheAlaValIleLeuGluAlaTyrLeuArg     675680685     GlyCysGlyThrAlaMetLeuHisAspPheThrGlnGlnValGlnVal     690695700     IleGluMetLeuGlnLysValThrLeuAspIleLysSerLeuSerAla     705710715720     GluLysTyrAspValSerSerGlnValIleSerGlnLeuLysGlnLys     725730735     LeuGluAsnLeuGlnAsnSerGlnLeuProGluSerPheArgValPro     740745750     TyrAspProGlyLeuLysAlaGlyAlaLeuAlaIleGluLysCysLys     755760765     ValMetAlaSerLysLysLysProLeuTrpLeuGluPheLysCysAla     770775780     AspProThrAlaLeuSerAsnGluThrIleGlyIleIlePheLysHis     785790795800     GlyAspAspLeuArgGlnAspMetLeuIleLeuGlnIleLeuArgIle     805810815     MetGluSerIleTrpGluThrGluSerLeuAspLeuCysLeuLeuPro     820825830     TyrGlyCysIleSerThrGlyAspLysIleGlyMetIleGluIleVal     835840845     LysAspAlaThrThrIleAlaLysIleGlnGlnSerThrValGlyAsn     850855860     ThrGlyAlaPheLysAspGluValLeuAsnHisTrpLeuLysGluLys     865870875880     SerProThrGluGluLysPheGlnAlaAlaValGluArgPheValTyr     885890895     SerCysAlaGlyTyrCysValAlaThrPheValLeuGlyIleGlyAsp     900905910     ArgHisAsnAspAsnIleMetIleThrGluThrGlyAsnLeuPheHis     915920925     IleAspPheGlyHisIleLeuGlyAsnTyrLysSerPheLeuGlyIle     930935940     AsnLysGluArgValProPheValLeuThrProAspPheLeuPheVal     945950955960     MetGlyThrSerGlyLysLysThrSerProHisPheGlnLysPheGln     965970975     AspIleCysValLysAlaTyrLeuAlaLeuArgHisHisThrAsnLeu     980985990     LeuIleIleLeuPheSerMetMetLeuMetThrGlyMetProGlnLeu     99510001005     ThrSerLysGluAspIleGluTyrIleArgAspAlaLeuThrValGly     101010151020     LysAsnGluGluAspAlaLysLysTyrPheLeuAspGlnIleGluVal     1025103010351040     TrpGlnArgGlnArgMetAspCysAlaVal     10451050     (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 15 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:     AsnSerGlnLeuProGluSerPheArgValProTyrAspProGly     151015     (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH:7 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:     LysAsnGlyAspAspLeuArg     15     (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH:5 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:     HisIleAspPheGly     15     __________________________________________________________________________ 

We claim:
 1. An isolated nucleic acid comprising:(a) a nucleotide sequence encoding an active phosphatidylinositol-3-kinase consisting of the amino acid sequence set forth in SEQ ID NO:2; (b) a nucleotide sequence encoding an active phosphatidylinositol-3-kinase consisting of the amino acid sequence set forth in SEQ ID NO:4; or (c) a nucleotide sequence which hybridizes with the nucleotide sequence of (a) or (b) after washing at 55° C. in an aqueous low salt buffer containing 0.2×SSC or 0.1% SDS.
 2. The nucleic acid of claim 1, wherein the nucleic acid is a recombinant DNA molecule.
 3. The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence consisting of:(a) the protein-coding sequence set forth in nucleotides 423-3569 of SEQ ID NO: 1; (b) the protein-coding sequence set forth in nucleotides 423-3572 of SEQ ID NO: 3; or (c) a sequence which hybridizes with the protein-coding sequence of (a) or (b) after washing at 55° C. in an aqueous low salt buffer containing 0.2×SSC or 0.1% SDS.
 4. The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence which hybridizes with the protein-coding sequence of (a) or (b) after washing at 62° C. in an aqueous low salt buffer containing 0.2×SSC or 0.1% SDS.
 5. The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence which hybridizes with the protein-coding sequence of (a) or (b) after washing at 68° C. in an aqueous low salt buffer containing 0.2×SSC or 0.1% SDS.
 6. An isolated nucleic acid comprising a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the nucleotide sequence is specific for nucleic acid encoding phosphatidylinositol-3-kinase γ.
 7. A vector, comprising the nucleic acid of one of claims 1-6.
 8. A transformed cell, containing the nucleic acid of one of the claims 1-6.
 9. A transformed cell, containing the vector of claim
 7. 10. A method for determining a phosphatidylinositol-3-kinase mRNA produced in cells, comprising incubating a nucleic acid probe comprising a phosphatidylinositol-3-kinase-mRNA-specific region of the nucleic acid of claim 1 with the phosphatidylinositol-3-kinase mRNA and determining any binding between the probe and the mRNA.
 11. The method of claim 10, wherein the cells are human cells. 